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. 2015 Mar 27;16(1):45.
doi: 10.1186/s12931-015-0206-6.

Megakaryoblastic leukemia-1 is required for the development of bleomycin-induced pulmonary fibrosis

Ksenija Bernau  1 , Caitlyn Ngam  2 , Elizabeth E Torr  3 , Benjamin Acton  4 , Jacob Kach  5 , Nickolai O Dulin  6 , Nathan Sandbo  7
Affiliations

Megakaryoblastic leukemia-1 is required for the development of bleomycin-induced pulmonary fibrosis

Ksenija Bernau et al. Respir Res. .

Abstract

Background: Fibrosing disorders of the lung, such as idiopathic pulmonary fibrosis, are characterized by progressive extracellular matrix accumulation that is driven by myofibroblasts. The transcription factor megakaryoblastic leukemia-1 (MKL1) mediates myofibroblast differentiation in response to several profibrotic stimuli, but the role it plays in mediating pulmonary fibrosis has not been fully elucidated. In this study, we utilized mice that had a germline deletion of MKL1 (MKL1 (-,-)) to determine the role that MKL1 plays in the development of bleomycin-induced pulmonary fibrosis.

Methods: Bleomycin or normal saline were intratracheally delivered to 9 to 12 week old female MKL1 (+,+) and MKL1 (-,-) mice. Mice were assessed for weight loss and survival to 28 days. Inflammatory responses were assessed through bronchoalveolar lavage at days 3 and 7 post-treatment. The development of pulmonary fibrosis was characterized using hydroxyproline assay and histological staining. MKL1 (+,+) and MKL1 (-,-) mouse lung fibroblasts were isolated to compare morphologic, gene expression and functional differences.

Results: MKL1 (-,-) mice demonstrated increased survival, attenuated weight loss, and decreased collagen accumulation compared to wild-type animals 28-days after intratracheal instillation of bleomycin. Histological analysis demonstrated decreased trichrome, smooth muscle α-actin, and fibronectin staining in MKL1(-,-) mice compared to MKL1 (+,+) controls. Differential cell counts from bronchoalveolar lavage demonstrated that there was attenuated neutrophilia 3 days after bleomycin administration, but no difference at day 7. Isolated mouse lung fibroblasts from MKL1 (-,-) mice had decreased contractility and deposited less fibronectin matrix compared to wild-type controls, suggesting a defect in key remodeling functions.

Conclusions: Altogether, these data demonstrate that MKL1 plays a significant role in mediating the fibrotic response to bleomycin injury. Loss of MKL1 attenuated early neutrophil influx, as well as myofibroblast-mediated remodeling. Targeting MKL1 activity may therefore be a useful strategy in treating pulmonary fibrosis.

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Figures

Figure 1
Figure 1
MKL1 (−,-) mice demonstrate increased survival and decreased collagen accumulation following experimentally-induced fibrosis compared to MKL1 (+,+) controls. A. Comparison of fraction of original weight over time (up to day 28) for MKL1 (+,+) and MKL1 (−,-) mice. B. Percent survival of MKL1 (+,+) and MKL1 (−,-) animals over time (up to day 28). Log-rank test (p < 0.05) was used to assess statistical significance. C. Quantitation of collagen accumulation in the left lung of MKL1 (+,+) and MKL1 (−,-) animals 28 days following saline or bleomycin treatment using hydroxyproline assay. D. Left lung dry weights of MKL1 (+,+) and MKL1 (−,-) animals treated with either normal saline or bleomycin. E. mRNA expression of MKL2, α-SMA, fibronectin and collagen1α1 in MKL1 (+,+) and MKL1 (−,-) untreated mouse lungs. One-way ANOVA (p < 0.05) was used to test for statistical significance.
Figure 2
Figure 2
MKL1 (−,-) mouse lungs following experimentally-induced fibrosis have decreased collagen, α-SMA and fibronectin accumulation compared to MKL1 (+,+) controls. A. Trichrome staining of MKL1 (+,+) and MKL1 (−,-) mouse lungs following bleomycin or saline treatment denoting areas of collagen accumulation (blue) with the corresponding modified Ashcroft score for each condition. B. Immunohistochemical staining for α-SMA (brown) and C. fibronectin (brown) including their respective quantitation. One-way ANOVA (p < 0.05) was used for statistical significance.
Figure 3
Figure 3
The early inflammatory response to bleomycin does not differ between MKL1 (+,+) and MKL1 (−,-) mice. A. Total BAL cell counts obtained 3 days following intratracheal saline or bleomycin treatment in MKL1 (+,+) and MKL1 (−,-) mice. B. Absolute neutrophil counts from the day 3 BALs obtained in Figure 3 A. C. Modified Ashcroft scores from trichrome-stained MKL1 (+,+) and MKL1 (−,-) mouse lungs done 3 days after bleomycin treatment. D. BAL differential cell counts of the same day 3 groups as in Figure 3 A, depicting the percentage of neutrophils, macrophages, lymphocytes and eosinophils counted following saline and bleomycin treatment in MKL1 (+,+) and MKL1 (−,-) mice. E. Total BAL cell counts obtained 7 days following intratracheal saline or bleomycin treatment in MKL1 (+,+) and MKL1 (−,-) mice. F. Absolute neutrophil counts from the day 7 BALs obtained in Figure 3 E. G. Modified Ashcroft scores from trichrome-stained MKL1 (+,+) and MKL1 (−,-) mouse lungs done 7 days after bleomycin treatment. H. BAL differential cell counts of the same day 7 groups as in Figure 3 E, depicting the percent of neutrophils, macrophages, lymphocytes and eosinophils counted following saline and bleomycin treatment in MKL1 (+,+) and MKL1 (−,-) mice. One-way ANOVA (p < 0.05) was performed to test for statistical significance.
Figure 4
Figure 4
MKL1 is required for contractile functions of the myofibroblast. A. Phase contrast microscopy of representative MKL1 (+,+) and MKL1 (−,-) primary mouse lung fibroblasts (MLF) grown on 2D plates (top row) and 3D in collagen (bottom row) to show morphological differences. B. MKL1 (+,+) and MKL1 (−,-) primary MLF lysates were analyzed by Western blot with antibodies against fibronectin (FN), vinculin, α-SMA and α-tubulin. Densitometry of n = 3 experiments (bottom panel). C. Merged images of 2D primary MLF subjected to immunofluorescent staining with phalloidin rhodamine (red) and or primary antibodies directed against vinculin and FITC-conjugated secondary antibodies (green). D. Cells stained by indirect immunofluorescence against vinculin were analyzed for focal adhesion size by quantitation of vinculin plaque length using ImageJ (as described in methods). E. MKL1 (+,+) and MKL1 (−,-) primary MLF were treated with 1 ng/ml TGF-β (72 hrs) or vehicle control prior to plating into 3D collagen gels for gel contraction assessment over 2 days. Gel areas were quantified from the digital images using ImageJ (right panel). F. MKL1 (+,+) and MKL1 (−,-) MLF were plated, serum-starved and stimulated with 1 ng/ml TGF-β or vehicle control for 24 hours. Cells were subjected to deoxycholate (DOC) extraction, with DOC-soluble and DOC-insoluble lysates subjected to gel electrophoresis under reducing (first panel) and non-reducing (second panel) conditions followed by Western blotting for total fibronectin. Fibronectin bands from both soluble and insoluble lysates were quantified by densitometry and values expressed as the ratio of insoluble to soluble fibronectin (third panel). One-way ANOVA (p < 0.05) was performed to test for statistical significance.
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